Characterization of the plasma membrane ATPase of Candida tropicalis

1) Plasma membrane vesicles from Candida tropicalis were isolated from protoplasts by differential centrifugation and purified in a continuous sucrose gradient. 2) The plasma membrane bound ATPase was characterized. It is highly specific for ATP and requires Mg2+. It is stimulated by K+, Na+ and NH4+. Lineweaver-Burk plots for ATPase activity are linear with a Vmax of 4.2 mumoles of ATP hydrolyzed min-1.mg-1 protein and a Km for ATP of 0.76 mM. The ATPase activity is inhibited competitively by ADP with a Ki of 1.7 mM and non competitively by vanadate with a Ki of 3 microM. The activity is unaffected by oligomycin or azide but is sensitive to DCCD.     

Mildly oxidized low-density lipoprotein inhibits the in vitro induction of the specific antibody response to Candida albicans

Oxidized low-density lipoprotein plays a critical role in the pathogenesis of atherosclerosis and exerts pleiotropic effects on various cellular functions. The present study was designed to evaluate the effects of mildly oxidized LDL (mLDL) on the induction and regulation of an in vitro specific antibody response. We found that mLDL significantly inhibited the induction of the anti-Candida albicans antibody response by human peripheral blood mononuclear cells (PBMC). mLDL-induced down-regulation of antibody production was abrogated by blocking the major receptors that bind and internalize modified LDL. In the mLDL-treated C. albicans-stimulated PBMC cultures an early increase in IL-1beta production was observed and the addition of anti-IL-1beta antibody abrogated the mLDL-induced inhibitory effect. Moreover, the addition of IL-1beta to the cultures inhibited the induction of the specific antibody response, similar to mLDL. On the other hand, mLDL up-regulated PWM-induced polyclonal immunoglobulin (Ig) production. In the same cultures IgM anti-mLDL was found. These results indicate that the up-regulation of IL-1beta production induced by mLDL may be involved in the hindering of B cell function, i.e., specific antibody production. This could be relevant in the pathogenesis of inflammatory diseases such as atherosclerosis.     

Iron–sulphur–phosphorus Interactions: Implications for Sediment Buffering Capacity in a Mediterranean Eutrophic Lagoon (Sacca di Goro,Italy)

The interactions among sedimentary cycles of sulphur, iron and phosphorus were investigated in the eutrophic Sacca di Goro lagoon (Northern Adriatic coast, Italy) in order to assess the iron buffering capacity of the sediment. Three stations were chosen, which represented different primary producer communities, hydrodynamics and sediment characteristics. Station G was close to the outlet of the Po di Volano river, station 4 was in the central part of the lagoon under tidal influence and station 17, in the sheltered zone, was affected by macroalgal blooms. From January 1997 to January 1998, sediment cores were sampled approximately every 2 months. In parallel, temperature, salinity and dissolved oxygen were determined in the water column. Sedimentary profiles of Eh, pH, dissolved sulphide (DS), acid volatile sulphide (AVS), chromium reducible sulphur (CRS), iron and phosphorus pools were determined in the 0–10 cm sediment horizon. Bacterial sulphate reduction rates were measured only at station 17. Iron pools, AVS and CRS followed similar patterns at all the considered stations, with lower values in the 0–2 cm sediment horizon and peaks in the deepest sediment layer (5–10 cm horizon). Overall, the labile Fe accounted for an annual average of 115.6 ± 3.0 μmol cm⁻³, with peaks of labile ferric iron up to 40 μmol cm⁻³. The great iron availability and reactivity accounted for an efficient buffering capacity against sulphides, with the accumulation of AVS and CRS, specially at station G where the iron buffer was replenished by iron-rich freshwaters. At station 17, in spite of a great iron availability the buffering capacity was less efficient due to macroalgal blooms and accumulation of labile organic matter, whose decomposition stimulated bacterial sulfate reduction and the inherent sulphide production both in the pore-water (DS: 4.0 ± 3.7 mM) and in the deepest water column (DS: 85 μM). The iron and sulphur interactions had also implications for P cycling, since in summer the ferric iron reduction was accompanied by a significant increase of pore-water and exchangeable phosphates. The AVS to labile Fe and AVS to TRIS (AVS + CRS) ratios were used to assess the sediment capacity of precipitating and retaining sulphides. These ratios were then compared with the chemical buffer capacity, demonstrating their suitability as indicators of buffering capacity towards sulphides.     

Platinum-assisted preparation of PS ligands containing chiral centres; X-ray crystal structure of a dithiophosphinite platinum complex

The design and synthesis of some ligands containing P(III) bonded to sulfur (thiophosphinites) and chiral centres is described in this paper.Their complexes with platinum (II), [PtCl₂L], (L = bidentate dithiophosphinite) have been prepared and characterised and it has been shown that in many cases, the coordination to platinum protects these ligands from decomposition processes operated by moisture and oxygen. The first example of X-ray crystal structure of a platinum coordinated dithiophosphinite is described for complex cis-[PtCl₂L], [L = meso-2,3-bis(diphenylthiophosphinito)-dimethyl-succinate], 4a.     

Leptin promotes differentiation and survival of human dendritic cells and licenses them for Th1 priming

Leptin is an adipocyte-derived hormone/cytokine that links nutrition, metabolism, and immune homeostasis. Leptin is capable of modulating several immune responses. However, the effect of leptin on dendritic cells (DCs) has not yet been recognized. Because DCs are instrumental in the development of immune responses, in this study, we evaluated the impact of leptin on DC activation. We demonstrated the presence of leptin receptor in human immature and mature DCs both at mRNA and protein level and its capacity to transduce leptin signaling leading to STAT-3 phosphorylation. We found no consistent modulation of DC surface molecules known to be critical for their APC function in response to leptin. In contrast, we found that leptin induces rearrangement of actin microfilaments, leading to uropod and ruffle formation. At a functional level, leptin up-regulates the IL-1beta, IL-6, IL-12, TNF-alpha, and MIP-1alpha production. Coincident with this, leptin-treated DCs stimulate stronger heterologous T cell responses. Furthermore, we found that leptin down-regulates IL-10 production by DCs and drives naive T cell polarization toward Th1 phenotype. Finally, we found that leptin partly protects DCs from spontaneous and UVB-induced apoptosis. Consistent with the antiapoptotic effect of leptin, we observed the activation of NF-kappaB and a parallel up-regulation of bcl-2 and bcl-x(L) gene expression. These results provide new insights on the immunoregulatory function of leptin demonstrating its ability to improve DC functions and to promote DC survival. This is of relevance considering a potential application of leptin in immunotherapeutic approaches and its possible use as adjuvant in vaccination protocols.     

Molecular organization and clustering of cell-wall-bound enzymes as a source of kinetic apparent co-operativity

When fixed charges and enzyme molecules are not homogeneously distributed in a matrix, the degree of organization of charges, of enzyme molecules and of charges with respect to enzyme molecules modulate the enzyme reaction rate. The overall reaction velocity of the bound enzyme system may be expressed in terms of monovariate moments of the charge density distribution and of the bivariate moments of the charge and enzyme density distributions. With respect to the situation where fixed charges and enzyme molecules are randomly distributed in the matrix, the molecular organization, as expressed by the monovariate and bivariate moments results in an increase or a decrease, of the overall reaction rate, as well as in the appearance of a kinetic cooperativity. The degree of spatial organization of objects may be expressed quantitatively through the concept of minimal spanning tree. This concept may thus be applied to the quantification of the degree of order that may exist in the bidimensional distribution of enzyme molecules in a charged matrix. Primary walls of isolated plant cells in sterile culture behave as a polyanion and contain different enzymes. The spatial distribution in sycamore cell walls of an acid phosphatase has been studied through the concept of minimal spanning tree and shown to be non-randomly distributed in the polyanionic matrix, but clustered in that matrix. This spatial organization results in a modulation of the reaction rate of the cell-wall-bound phosphatase reaction. Both the theoretical and experimental results presented in this study leave little doubt as to the validity of the idea that in situ the organization of fixed charges and enzyme molecules modulate the overall dynamics of enzyme reactions.     

Lotus corniculatus nodulation specificity is changed by the presence of a soybean lectin gene

Plant lectins have been implicated as playing an important role in mediating recognition and specificity in the Rhizobium-legume nitrogen-fixing symbiosis. To test this hypothesis, we introduced the soybean lectin gene Le1 either behind its own promoter or behind the cauliflower mosaic virus 35S promoter into Lotus corniculatus, which is nodulated by R. loti. We found that nodulelike outgrowths developed on transgenic L. corniculatus plant roots in response to Bradyrhizobium japonicum, which nodulates soybean and not Lotus spp. Soybean lectin was properly targeted to L. corniculatus root hairs, and although infection threads formed, they aborted in epidermal or hypodermal cells. Mutation of the lectin sugar binding site abolished infection thread formation and nodulation. Incubation of bradyrhizobia in the nodulation (nod) gene-inducing flavonoid genistein increased the number of nodulelike outgrowths on transgenic L. corniculatus roots. Studies of bacterial mutants, however, suggest that a component of the exopolysaccharide surface of B. japonicum, rather than Nod factor, is required for extension of host range to the transgenic L. corniculatus plants.     

Characterization of the chromosome complement of Helianthus annuus by in situ hybridization of a tandemly repeated DNA sequence.

A tandemly repeated sequence isolated from a clone (HAG004N15) of a nebulized genomic DNA library of sunflower (Helianthus annuus L., 2n = 34) was characterized and used to study the chromosome complement of sunflower. HAG004N15 repeat units (368 bp in length) were found to be highly methylated, and their copy number per haploid (1C) genome was estimated to be 7800. After in situ hybridization of HAG004N15 repeats onto chromosome spreads, signals were observed at the end of both chromosome arms in 4 pairs and at the end of only one arm in 8 other pairs. Signals were also observed at the intercalary (mostly subtelomeric) regions in all pairs, in both arms in 8 pairs, and in only one arm in the other 9 pairs. The short arm of 1 pair was labelled entirely. The chromosomal location of ribosomal DNA was also studied by hybridizing the wheat ribosomal probe pTa71. Four chromosome pairs contained ribosomal cistrons at the end of their shorter arm, but a satellite was seen in only 3 pairs. These hybridization patterns were the same in the 3 sunflower lines studied (HA89, RA20031, and HOR). The chromosomal localization of HAG004N15-related sequences allowed all of the chromosome pairs to be distinguished from each other, in spite of small size and similar morphology.     

The Brazilian Amaryllidaceae as a source of acetylcholinesterase inhibitory alkaloids

Nine Brazilian Amaryllidaceae species were studied for their alkaloid composition and acetylcholinesterase (AChE) inhibitory activity via GC–MS and a modified Ellman assay, respectively. A total of thirty-six alkaloids were identified in these plants, of which Hippeastrum papilio and H. glaucescens exhibited the highest galanthamine content and the best IC₅₀ values against AChE. Furthermore, Hippeastrum vittatum and Rhodophiala bifida also showed notable AChE inhibitory effects. X-ray crystallographic data for four galanthamine-type compounds revealed significant differences in the orientation of the N-methyl group, which are shown to be related to AChE inhibition.